CN115177600A - Protein phospholipid nanometer preparation, application and preparation method thereof - Google Patents
Protein phospholipid nanometer preparation, application and preparation method thereof Download PDFInfo
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- CN115177600A CN115177600A CN202210510621.9A CN202210510621A CN115177600A CN 115177600 A CN115177600 A CN 115177600A CN 202210510621 A CN202210510621 A CN 202210510621A CN 115177600 A CN115177600 A CN 115177600A
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Abstract
The invention provides a protein phospholipid nanometer preparation, application and a preparation method thereof. Wherein the protein phospholipid nano preparation is a protein phospholipid complex based on combination of ApoA1 protein and phospholipid; the ApoA1 protein is ApoA1 protein which does not contain a conserved sequence shown in SEQ ID No.1 or a homologous sequence of the conserved sequence. The protein phospholipid complex provided by the invention can be used as a drug for resisting CNS diseases and a brain targeted delivery system, and becomes a solution of a brain drug delivery system for preventing and treating CNS diseases.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a protein phospholipid nano preparation, application and a preparation method thereof.
Background
Diseases of the Central Nervous System (CNS) seriously affect human health, for example, neurodegenerative diseases (e.g. alzheimer's disease, parkinson's disease), meningitis, brain tumors (e.g. gliomas, central neurolymphomas, etc.).
Alzheimer's Disease (AD) is one of the most common neurodegenerative diseases, with approximately 10-12% of the population 65 years and older being affected by AD. The AAIC international conference held in 2021 promulgated a number of reports (World Alzheimer Report 2021) with an expected total of 1.52 billion patients with about 60-70% Alzheimer's disease patients by 2050. At present, 1000 million Alzheimer disease patients exist in China, the number of the Alzheimer disease patients is the first of the whole world, and the number of the Alzheimer disease patients can reach 4000 million people by 2050. As the population of china ages, alzheimer's disease, as well as other forms of dementia and major neurocognitive disorders, are increasing dramatically. And the prevalence rate of Parkinson's Disease (PD) in china above 65 years is currently about 1.7%.
The Blood Brain Barrier (BBB) severely limits the treatment of Central Nervous System (CNS) diseases (e.g., neurodegenerative diseases, brain tumors, brain infections, and stroke) because it prevents 98% of small molecule drugs and large molecules (e.g., peptides, gene drugs, and protein drugs) from entering the brain.
Therefore, aiming at the bottleneck of the current Central Nervous System (CNS) diseases, the development of a protein system which can efficiently pass through a blood brain barrier, has high brain targeting property, is safe and nontoxic, and effectively prevents early neurodegenerative diseases and the like is urgently needed.
Disclosure of Invention
In order to solve the problems, the invention provides a protein phospholipid nano preparation which is a protein phospholipid compound based on combination of ApoA1 protein and phospholipid;
wherein, the ApoA1 protein is ApoA1 protein without conserved sequence shown in SEQ ID No.1 or homologous sequence of the conserved sequence.
Preferably, the amino acid sequence of the protein phospholipid nano-formulation comprises:
one or two of 1 st amino acid sequence shown as SEQ ID No.2 and 2 nd amino acid sequence shown as SEQ ID No. 3.
Preferably, the amino acid sequence of the protein phospholipid nano-formulation further comprises:
the 3 rd amino acid sequence shown as SEQ ID No. 3.
Preferably, the sequence of the amino acid sequence of the protein phospholipid nanometer preparation is that the 1 st amino acid sequence, the 2 nd amino acid sequence and the 3 rd amino acid sequence are arranged in sequence.
Preferably, each nanoparticle of the proteolipid nano-formulation has a diameter of 5-130nm.
Preferably, the number of phospholipids embedded inside each nanoparticle of the protein phospholipid nano-formulation is 100-1000.
Preferably, the protein phospholipid nanometer preparation is applied to the preparation of products for preventing and/or treating early neurodegenerative diseases.
Preferably, the use of the nano-formulation of protein phospholipids for the preparation of a product for the treatment and/or prevention of CNS diseases.
In addition, in order to solve the above problems, the present application also provides a brain drug delivery system comprising the protein phospholipid nano-formulation as described above.
In addition, in order to solve the above problems, the present application also provides a method for preparing the above protein phospholipid nano-preparation, comprising:
taking ApoA1 protein, and obtaining a crude protein sample after induced expression;
purifying the crude protein sample to obtain purified protein;
and assembling the purified protein and dimyristoyl phosphatidylcholine to obtain the protein phospholipid nanometer preparation.
The invention provides a protein phospholipid nanometer preparation, application and a preparation method thereof. The protein phospholipid nano preparation is a protein phospholipid compound which is designed by using genetic engineering and based on the combination of ApoA1 protein and phospholipid, and has the following beneficial effects:
1. the protein phospholipid nanometer preparation can effectively penetrate through a blood brain barrier on one hand, and can obviously show a remarkable treatment effect on early neurodegenerative diseases (including AD diseases) on the other hand.
2. The phospholipid in the protein phospholipid nanometer preparation belongs to natural phospholipid, and the ApoA1 protein has good biocompatibility, so that the protein phospholipid nanometer preparation can achieve better stability in vivo;
3. the protein phospholipid nanometer preparation can load corresponding micromolecules and can be used as a drug carrier.
In conclusion, the protein phospholipid nanometer preparation can be used as a medicine for resisting CNS diseases, can also be used as a brain targeting delivery system, and becomes a solution of the brain medicine delivery system for preventing and treating CNS diseases.
Drawings
FIG. 1 is a schematic ribbon diagram of ApoA1 protein containing conserved sequences;
FIG. 2 is a SDS-PAGE pattern of ApoA1 protein after purification;
FIG. 3a is a Size Exclusion (SEC) plot of ApoA 1-ND;
FIG. 3 (b-1) is a Transmission Electron Microscope (TEM) image of ApoA1-ND for large size particles (125 nm);
FIG. 3 (b-2) is a Transmission Electron Microscope (TEM) image of ApoA1-ND for medium size particles (38 nm);
FIG. 3 (b-3) is a Transmission Electron Microscope (TEM) image of ApoA1-ND for small size particles (4.6 nm);
FIG. 3 (c-1) is an atomic force microscope nanoparticle morphology map;
FIG. 3 (c-2) is a height diagram of atomic force microscope nanoparticles;
FIG. 4 is a brain profile following intravenous administration of Bodipy-labeled ApoA 1-ND;
FIG. 5 is a graph of accumulation of ApoA1-ND around A β aggregates following intravenous injection;
FIG. 6 is a photograph of a section of brain tissue with ApoA1-ND reducing A β deposition;
FIG. 6 (a) shows that ApoA1-ND reduces amyloid deposition in cerebral cortex and hippocampus of APP/PS1 mice;
FIG. 6 (b) is a statistical plot of the quantitative analysis of amyloid deposition in cerebral cortex of APP/PS1 mice;
FIG. 6 (c) is a statistical chart of the quantitative analysis of amyloid deposition in the hippocampus of APP/PS1 mice;
FIG. 7 is a photograph of a section of brain tissue with ApoA1-ND reducing microglial proliferation;
FIG. 7 (a) shows that ApoA1-ND reduces attenuated microglial proliferation in cerebral cortex and hippocampus of APP/PS1 mice;
FIG. 7 (b) is a statistical plot of the quantitative analysis that ApoA1-ND reduces microglial proliferation in cerebral cortex of APP/PS1 mice;
FIG. 7 (c) is a statistical plot of the quantitative analysis that ApoA1-ND reduces microglial proliferation in the hippocampus of APP/PS1 mice;
FIG. 8 is a graph showing the results of Nicol staining of tissue sections;
fig. 9 (a) is a time table of behavioral trace analysis (morse water maze experiment) treatment, pathology monitoring and treatment assessment of alzheimer model mice;
FIG. 9 (b) is a graph of the escape latency at different time points;
FIG. 9 (c) is a graph comparing swimming speeds;
FIG. 9 (d) is the number of times the mouse passed over the platform on the last day of platform removal;
FIG. 9 (e) is a representative swimming path of MWM mice after different treatments;
FIG. 10 is a sectional view of each organ tissue.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of 8230A" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.
Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.
The term "about" in the present invention denotes an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The following is provided merely to aid in understanding the invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.
The technical solution of the present invention is further described in detail by way of the following specific embodiments, but the present invention is not limited thereto, and any limited number of modifications made by anyone within the scope of the claims of the present invention are still within the scope of the claims of the present invention.
The invention provides a protein phospholipid nanometer preparation, which is a protein phospholipid compound based on combination of ApoA1 protein and phospholipid; wherein, the ApoA1 protein is ApoA1 protein without conserved sequence shown in SEQ ID No.1 or homologous sequence of the conserved sequence.
Above, apoA1 is a main component of high density lipoprotein, can be assembled into phospholipid nanodiscs as a scaffold of phospholipid nanodiscs, and is an apolipoprotein.
The periphery of the phospholipid nanodisk is wrapped by amphiphilic membrane scaffold protein, so that a stable bilayer membrane structure can be formed, the structure composition is stable, and the phospholipid nanodisk is not influenced by the change of phospholipid concentration.
As described above, phospholipids, which are natural phospholipids in the present invention, have very good stability and biocompatibility. It should be noted that, according to the literature (Human apolipoprotein A-I binding analog-beta and preceding A beta-induced neurovirulence, INT J BIOCHEM CELL B,41 (2009) 1361-1370), the conserved sequence "GNLLTLD" fragment, which has homology with ApoA1 protein, and the sequence GNLLTLD and its homologous sequence can bind specifically with aggregated A beta, prove that the homologous sequence is the binding site of A beta. Referring to fig. 1, the spatial band structure of ApoA1 protein containing conserved sequence segments is shown. Table 1 shows the homologous sequences with similar fragments to the conserved sequence, and the fragments from sequences 2 to 18 have the same "GNLLTLD" as the "GNLLTLD" fragment in conserved sequence 1, i.e., the homologous sequences (shown in bold and italic letters). For example, "LNLKLLD" in SEQ ID No.2 has corresponding segments "NL" and "LD" identical to the conserved sequence, but which are homologous sequences similar to the conserved sequence, although not identical.
TABLE 1 homologous sequences with similar fragments to conserved sequences
Conventional ApoA 1-related proteins of high-density lipoproteins have fragments containing the above-mentioned homologous sequences, i.e., conserved sequences, so that the ApoA 1-related proteins can target a β and promote a β degradation after crossing the blood-brain barrier.
The protein phospholipid nanometer preparation provided by the invention is a protein phospholipid complex combined by ApoA1 protein and phospholipid. As one of ApoA 1-related proteins, there is a fragment interval in which the same conserved sequence as in ApoA 1-related protein of conventionally disclosed high-density lipoprotein is not contained, but exhibits an interaction ability with a β, and particularly, a significant therapeutic effect on early neurodegenerative disease AD disease, which has not been reported in the literature.
The protein phospholipid nanometer preparation provided by the invention can target Abeta through a Blood Brain Barrier (BBB) and promote the degradation of Abeta, reduce the aggregation level of Abeta, reduce the proliferation of microglial cells, reduce the loss of hippocampus, namely cerebral cortical neurons, improve the spatial learning and memory capacity, and can show remarkable early neurodegenerative disease prevention and treatment effects.
The protein phospholipid nanometer preparation provided by the invention can effectively cross a blood brain barrier on one hand, and can obviously show a remarkable treatment effect on early neurodegenerative diseases (including AD diseases) on the other hand; the phospholipid in the protein phospholipid nanometer preparation belongs to natural phospholipid, and the ApoA1 protein has good biocompatibility, so that the protein phospholipid nanometer preparation can achieve better stability in vivo; the protein phospholipid complex can load corresponding micromolecules and can be used as a drug carrier; the protein phospholipid complex can be used as a medicine for resisting CNS diseases, can also be used as a brain targeting delivery system, and becomes a solution of a brain medicine delivery system for preventing and treating CNS diseases.
Further, the amino acid sequence of the protein phospholipid nanometer preparation comprises:
one or two of the 1 st amino acid sequence shown as SEQ ID No.2 and the 2 nd amino acid sequence shown as SEQ ID No. 3.
The amino acid sequence of the protein phospholipid nano preparation is ApoA1 protein expressed and modified in E.coli BL21 (DE 3), and the protein phospholipid nano preparation at least contains one or two of the 1 st amino acid sequence (a) and the 2 nd amino acid sequence (b).
Here, the protein phospholipid nano-preparation may include the following cases, which may be respectively: 1. a; 2. b; 3. a + b. Here again, the sequence between sequences a and b is not limited to the order of arrangement.
Further, the amino acid sequence of the protein phospholipid nanometer preparation also comprises:
the 3 rd amino acid sequence shown as SEQ ID No. 3.
Further, the sequence of the amino acid sequence of the nano preparation of the protein phospholipid is that the 1 st amino acid sequence, the 2 nd amino acid sequence and the 3 rd amino acid sequence are sequentially arranged.
In the expression of the modified ApoA1 protein in e.coli bl21 (DE 3), i.e. in the protein phospholipid nano-formulation, the sequence in the following order is included: said 1 st amino acid sequence, said 2 nd amino acid sequence, and said 3 rd amino acid sequence are arranged in order.
Further, each nanodisk particle of said proteolipid nanoformulation has a diameter of 5-130nm.
In the above protein phospholipid nano-preparation, after the ApoA1 protein conforms to the natural phospholipid, the particle size can be kept fixed, and the particle size specification can be as follows: the particle size can be maintained between 5 and 130nm.
Preferably, the number of phospholipids embedded inside each particle of the protein phospholipid nano-formulation is 100-1000.
As described above, apoA1 protein can also show good stability in a series of in vivo experiments, and is an ideal carrier platform for drug delivery applicable to imaging diagnosis and therapy, and after being combined with phospholipid, lipid molecules with the number of 100-1000 can be embedded into each nanoparticle body to form a complex, so as to form a complex biomacromolecule system.
Further, the protein phospholipid nanometer preparation is applied to preparing products for preventing and/or treating early neurodegenerative diseases.
Further, the application of the protein phospholipid nanometer preparation in preparing products for treating and/or preventing CNS diseases.
In addition, in order to solve the above problems, the present application also provides a brain drug delivery system, including the above-described proteolipid nano-formulation.
Drug Delivery System (DDS) refers to a technical System that regulates the distribution of drugs in vivo in space, time, and dosage in a comprehensive manner. The aim is to deliver a proper amount of drug to the right position at the right time, thereby increasing the utilization efficiency of the drug, improving the curative effect, reducing the cost and reducing the toxic and side effects. The drug delivery system is a fusion subject of medicine, engineering (material, mechanical, electronic) and pharmacy, and its research objects include not only the drug itself, but also a carrier material and device carrying the drug, and a related technology for performing physicochemical modification and modification on the drug or the carrier. In the application, a brain drug delivery system is provided, and relates to a drug delivery system with certain treatment and prevention functions for brain diseases.
The invention relates to a protein phospholipid nano preparation based on ApoA1 protein designed by genetic engineering, which is a carrier platform for drug delivery, and 100-1000 ApoA1 protein nano particles can be embedded after the protein phospholipid nano preparation is combined with phospholipid. The lipid molecules form a compound to form a compound biomacromolecule system, thereby realizing the carrier function of the drug delivery system, being used as a drug brain targeted delivery system for resisting CNS diseases, and becoming a set of solution for the drug brain delivery system for treating CNS diseases.
In addition, in order to solve the above problems, the present application also provides a method for preparing the above-mentioned nano preparation of protein phospholipid, comprising:
taking ApoA1 protein, and obtaining a crude protein sample after induced expression;
purifying the crude protein sample to obtain purified protein;
and assembling the purified protein and dimyristoyl phosphatidylcholine to obtain the protein phospholipid nanometer preparation.
In the assembling of the purified protein and dimyristoyl phosphatidylcholine, the molar ratio of the purified protein to the dimyristoyl phosphatidylcholine may be 1: (50-150).
Further, the molar ratio of the purified protein to dimyristoyl phosphatidylcholine may be 1: (60-120).
The invention is further illustrated by the following specific examples, but it should be understood that these examples are included merely for purposes of illustration in more detail and are not intended to limit the invention in any way.
Example 1: inducible expression of ApoA1 protein.
The preparation method comprises the following steps: and (3) taking ApoA1 protein, and obtaining a crude protein sample after induction expression.
The experimental method comprises the following steps:
(1) Taking a recombinant plasmid containing an ApoA1 protein gene sequence, and transforming the recombinant plasmid into an escherichia coli competent BL21 (DE 3) strain;
(2) After overnight culture, single clones were picked up in 50mL LB medium containing 100. Mu.g/mL kanamycin, placed on a shaker at 220r/min, and cultured overnight at 37 ℃.
(3) The inoculum was transferred to 1000mL LB medium containing 100 μ g/mL kanamycin to achieve initial OD600= 0.1. Shaking culture was carried out at 37 ℃ until OD600=0.8, and 1mL of IPTG was added to give a final concentration of 1mM.
(4) Placing on a shaking bed, and inducing at 37 deg.C and 220r/min for 4h. And taking a bacterium solution (crude protein sample) before and after induction, and storing the bacterium solution for subsequent SDS-PAGE gel electrophoresis.
Example 2: and (4) purifying the protein.
The preparation method comprises the following steps: and purifying the crude protein sample to obtain purified protein.
The experimental method comprises the following steps:
(1) Induced bacteria (crude protein samples) were collected by centrifugation, washed 2 times with pre-cooled TBS buffer, supernatant removed, lysate 20mM Tris-HCl pH =8.0,1% Triton X added to the pellet, cell mass resuspended thoroughly, and 1mg lysozyme added. Bacteria were lysed by low temperature ultrasound (ice bath). The lysate was centrifuged at 12000rpm for 45min at 4 deg.C, the supernatant collected and filtered, and then purified by Ni-NTA affinity column.
The buffers used were as follows: loading buffer solution: 1) pH =8.0,20mM Tris-HCl,500mM NaCl,1% TritonX (loading); 2) pH =8.0,20mM Tris-HCl,500mM NaCl,50mM Na-cholate (flush); elution buffer: 3) pH =8.0,20mM Tris-HCl,500mM NaCl,20mM imidazole (deproteinized); 4) pH =8.0,20mM Tris-HCl,500mM NaCl,50mM imidazole (trash protein); 5) pH =8.0,20mM Tris-HCl,500mM NaCl,500mM imidazole (elution of the protein of interest);
(2) After elution of the target protein, the column was dialyzed overnight against 20mM Tris-HCl,50mM NaCl buffer, pH = 8.0. Then, purification was continued by using an anion exchange column. Elution was performed with different concentrations of NaCl dissolved in Tris-HCl, pH =8.0,10 mM. A small amount of the eluted target protein was subjected to SDS-PAGE, and the remainder was dialyzed overnight against 20mM PBS buffer pH = 7.4.
(3) The single ApoA1 protein can be obtained after purification by nickel ion affinity chromatography and anion exchange chromatography.
The experimental results are as follows: referring to FIG. 2, the successful purification of the protein was shown by SDS-PAGE results. It should be noted that the ApoA1 yield in this example can be adjusted to a wide range according to actual needs. Wherein, the yield of the ApoA1 protein is that 1000mg of target protein can be obtained by expression and purification of every 100L of liquid culture medium.
Example 3: assembly of DMPC with ApoA1 protein.
The preparation method comprises the following steps: and assembling the purified protein and dimyristoyl phosphatidylcholine to obtain the protein phospholipid nanometer preparation.
The experimental method comprises the following steps: (1) Dimyristoyl phosphatidylcholine (DMPC) was dispersed in PBS buffer (20mM pH 7.4) containing sodium cholate; (2) The ApoA1 protein solution was added dropwise to DMPC and the experiment was performed in ultrasound until the solution became clear, with a molar ratio of ApoA1 protein to DMPC of 1. (3) The ApoA1 protein and DMPC mixture was dialyzed overnight against physiological saline and the supernatant was centrifuged for Size Exclusion Chromatography (SEC), transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) characterization.
The experimental results are as follows: referring to the AFM graphs of FIG. 3 (c-1) and FIG. 3 (c-2), the sizes of the individual proteolipid nanoformulation nanoparticles after assembly were measured in the AFM results, see the following table. The TEM result shows that the biological macromolecular system assembled by mixing ApoA1 protein and DMPC, namely the protein phospholipid nano preparation (hereinafter referred to as ApoA 1-ND) has the diameter of about 10nm, and the successful construction of the protein phospholipid complex ApoA1-ND is verified by the SEC graph of figure 3 (a) and the peak time of SEC, and the successful assembly of ApoA1-ND is proved.
Materials and apparatus in examples 4-11:
1. experimental materials: (1) Male BALB/C nude mice, 4-5 weeks old, 20 + -2 g. Purchased from beijing huafukang biotech science and technology gmbh. (2) Male APP/PS1 double transgenic mice, 7 months of age, 30 + -2 g. Purchased from Beijing Huafukang Biotech GmbH. (3) Male C57BL/6J mice, 7 months of age, 30. + -.2 g. Purchased from beijing huafukang biotech science and technology gmbh.
2. Experimental reagent: bodipy, saline, GSH, rabbit anti-APP polyclonal antibody, rabbit anti-PECAM 1 polyclonal antibody, alexaFluor 488-labeled goat anti-rabbit IgG, 4% paraformaldehyde, embedded paraffin, citric acid, methanol, hydrogen peroxide, triton X-100, concentrated normal goat serum (blocked), rabbit anti-PTRPC polyclonal antibody, immunohistochemical kit, nissl stain, absolute ethanol, xylene, hematoxylin, hydrochloric acid, neutral gum, dako REAL EnVision Detection System, toluidine blue, eosin Y (water soluble), anti-fluorescence quenching encapsulated tablet.
3. An experimental instrument: a dehydrator, an ultrapure water system, a pathological microtome, a slicing knife, a tissue spreading and baking machine, a glass slide and a cover glass, an electric ceramic furnace for antigen retrieval, a microscope and an embedding machine.
Animal feeding methods in examples 4-11: the experimental adopted mice are SPF mice, and the feeding temperature of all the mice is controlled to be 25 +/-1 ℃; the experimental mice can obtain clean granulated feed and sterile water at any time; the pad, squirrel cage material and drinking water were changed every 5 days.
Example 4: in vivo fluorescence imaging analysis of ApoA1-ND.
Purpose of the experiment: to determine that the solution of the protein phospholipid complex ND was able to cross the blood brain barrier into the center after intravenous administration, in vivo fluorescence imaging analysis was performed using fluorescent Bodipy labeled ApoA1-ND.
The experimental method comprises the following steps: 12 nude mice were randomly divided into two groups, and 200. Mu.L of physiological saline and Bodipy-Labeled ApoA1-ND were injected through tail vein, respectively. Image acquisition was performed at 15min, 1h, 4h, 10h and 20h after intravenous administration.
The experimental results are as follows: (1) 15 minutes: the image acquisition results at 15min of solution injection showed that the saline-only control group was non-fluorescent and Bodipy-labeled ApoA1-ND was mainly concentrated in the tail. (2) 1 hour: 1h image acquisition shows that the Bodipy marked ApoA1-ND enters blood circulation, enters the heart after entering the inferior vena cava, reaches the brain through the aorta and all branches thereof, can penetrate the blood brain barrier to enter the brain, and the fluorescence spreads all over the brain. (3) 4 hours: after 4h, the blood circulates through the aorta and branches of all levels to reach all parts of the whole body, the amount of ApoA1-ND reaches the maximum, and the amount of ApoA1-ND pumped into the brain is also greatly increased. (4) 10 hours: after 10h, the fluorescence intensity gradually decreased as the mice were metabolized.
To summarize: referring to fig. 4, as seen from the trend of the in vivo fluorescence imaging of ApoA1-ND at 15 minutes, 1 hour, 4 hours and 10 hours, apoA1-ND can be clearly shown to have the ability to effectively cross the BBB.
Example 5: a β immunofluorescence assay.
Purpose of the experiment: to demonstrate that ApoA1-ND can enter brain parenchyma following tail vein administration, we used an immunofluorescence technique in conjunction with confocal microscopy analysis to assess the ability of ApoA1-ND to target a β in vivo using an a β immunofluorescence assay.
The experimental method comprises the following steps: 1) 200 μ L of Bodipy-labeled ApoA1-ND tail vein at 20 μ M concentration was injected into APP/PS1 mice; 2) After 4 hours, mice were treated and two groups of frozen brain sections were prepared. To determine whether ApoA1-ND was delivered to brain parenchyma and bound to a β aggregates in the brain, a first set of sections labeled capillary blood with rabbit anti-PECAM 1 polyclonal antibody (primary antibody) and a second set labeled brain a β aggregates with rabbit anti-APP polyclonal antibody (primary antibody). 3) Taking out the frozen section from the refrigerator, recovering to room temperature, and soaking in PBS for several minutes; 4) Serum blocking: wiping the slide with absorbent paper, circling the tissue with an immune group painting brush, dripping diluted normal goat serum, and sealing at room temperature for 30min to reduce non-specific staining; 5) Adding a primary antibody: throwing off excessive liquid, not washing, then dropwise adding diluted primary antibody, and incubating overnight in a wet box at 4 ℃ after the primary antibody is added; 6) Secondary antibody addition (with fluorescence): washing the slices with PBST for 3 times, each time for 3min, wiping the slices with absorbent paper, dripping diluted fluorescent secondary antibody, incubating for 1h at 37 ℃ in a wet box, washing the slices with PBST for 4 times, each time for 3min, and performing the steps and all the following operation steps in a dark place as much as possible; 7) Counterstaining the nucleus: dripping DAPI, incubating for 5min in dark, staining the specimen for nucleus, and washing off excessive DAPI 5min × 4 times by PBST; 8) The liquid on the section is wiped dry by absorbent paper, the section is sealed by mounting liquid containing an anti-fluorescence quencher, and then the image is observed and collected under a fluorescence microscope.
The experimental results are as follows: referring to fig. 5, immunofluorescence signals (highlight positions of panels a β) by rabbit anti-APP polyclonal antibody show that in the major sites of a β aggregation, i.e., cerebral cortex (Cotex) and Hippocampus (Hippocampus), bodipy-labeled ApoA1-ND (highlight positions of panels ApoA 1-ND) are highly aggregated around a β.
To summarize: after 4h of tail vein administration, bodipy-labeled ApoA1-ND (red) was highly accumulated around a β in the cerebral cortex and hippocampus, which are major sites of a β accumulation. The result shows that ApoA1-ND can definitely target A beta deposition after passing through cerebral vessels to enter brain parenchyma.
Example 6: anti-a β immunostaining experiments.
Purpose of the experiment: to evaluate the targeting of ApoA1-ND to A β and its disease modifying effects in AD.
The experimental method comprises the following steps: male APP/PS1 mice of 7 months of age were divided into three groups of three mice each, and tail vein injections were performed separately: physiological saline (NS), 20. Mu.M ApoA1-ND, 5. Mu.M ApoA1-ND, injected in 200. Mu.L for 2 consecutive weeks, with C57BL/6J male mice of the same conditions as normal controls, injected with physiological saline (NS) alone, grouped as in the following table, two weeks later, mice were anesthetized, and the heart was perfused with cold saline and brain tissue was collected.
| Group of | Test procedure |
| 1(Control) | C57BL/6J mice + intravenous saline, continuous 2 |
| 2 | APP/PS1 mice + intravenous saline for 2 |
| 3 | APP/PS1 mice + intravenous ApoA1-ND (20. Mu.M) for 2 |
| 4 | APP/PS1 mice + intravenous ApoA1-ND (5. Mu.M) for 2 weeks |
The operation process comprises the following steps: tissue dehydration, tissue clearing, waxing, embedding, slicing and baking, slicing dewaxing, antigen repairing, endogenous peroxidase blocking, serum sealing, primary antibody adding, enzyme labeled secondary antibody adding, color developing agent adding, counterstaining, dehydration, mounting and microscopic examination photographing.
The experimental results are as follows: as can be seen in FIG. 6 (a), apoA1-ND reduced amyloid deposition (brown plaques) in the cerebral cortex and hippocampus of APP/PS1 mice. When the ApoA1-ND concentration is increased from 5 mu M to 20 mu M, the inhibition effect on the amyloid plaque amount is obviously enhanced, and the amyloid deposition amount is obviously reduced. As can be seen in FIG. 6 (b), apoA1-ND reduced amyloid deposition in cerebral cortex of APP/PS1 mice (statistical plot). As can be seen in FIG. 6 (c), apoA1-ND reduced amyloid deposition in the hippocampus of APP/PS1 mice (statistical plot).
In summary, the anti-a β immunostaining of fig. 6 (a), 6 (b) and (c) can clearly show a significant reduction in amyloid plaque burden in cortex and hippocampus of ApoA1-ND treated APP/PS1 double transgenic mice compared to saline treated APP/PS1 double transgenic mice.
Example 7: microglial activation evaluation experiment.
The purpose of the experiment is as follows: abnormal activation of microglia was observed in AD patients and in amyloidosis mouse models. Previous studies have shown that both Α β oligomers and fibrils can trigger the neuroinflammatory cascade. In this example, the activation of microglia was evaluated using PTRPC as a marker.
The experimental method comprises the following steps: 7 month old APP/PS1 mice (n =8-9 per group) received ApoA1-ND treatment, 200 μ L of 5 μ M ApoA1-ND, daily by tail vein injection for 2 weeks, using age-matched APP/PS1 and C57BL/6J mice given physiological saline as negative and normal controls, respectively. Brain sections (4 μm) were immunostained with rabbit anti-PTRPC polyclonal antibody.
The experimental results are as follows: as can be seen from FIG. 7 (a), apoA1-ND reduced attenuated microglial proliferation (brown signal) in cerebral cortex and hippocampus of APP/PS1 mice. As can be seen from FIG. 7 (b), apoA1-ND reduced microglial proliferation in cerebral cortex of APP/PS1 mice (statistical). As can be seen in FIG. 7 (c), apoA1-ND reduced microglial proliferation in the hippocampus of APP/PS1 mice (statistical plot).
In conclusion, apoA1-ND reduced amyloid (a β) deposition in different tissues of mice in the alzheimer model, and a significantly reduced burden of positively activated microglia in ApoA1-ND treated mice, compared to APP/PS1 double transgenic mice treated with saline.
In view of the central role of a β aggregates in AD brain microglial activation, the significant reduction in activated microglia observed in ApoA1-ND treated mice may be attributed to the activity of ApoA1-ND in promoting a β clearance.
Example 8: nissl staining analysis: observation of neuronal state of cortex and hippocampus.
Purpose of the experiment: neuronal loss in the cortex and hippocampus is one of the major features of AD. Therefore, the treatment effect of ApoA1-ND can be reflected by observing the neuron states of the cortex and hippocampus.
The experimental method comprises the following steps: the brain was examined for histological changes using Nissl staining.
1) Tissue dehydration; 2) The tissue is transparent; 3) Wax dipping; 4) Embedding; 5) Slicing and baking; 6) Slicing and dewaxing; 7) The slices are dyed in toluidine blue dye liquor (Nishi dye liquor) preheated to 60 ℃ and 1 percent for 40min, and washed by distilled water for 3 times; 8) Quickly differentiating by 95% alcohol, decoloring, and microscopically examining until the background is clear; the absolute ethyl alcohol is dehydrated quickly, the dimethylbenzene is transparent, and the neutral gum is sealed after the neutral gum is dried in a fume hood.
The experimental results are as follows:
as shown by Nissl staining analysis in FIG. 8, both cortical and hippocampal neuronal subcellular depletion and neuronal nuclear atrophy were observed in saline treated APP/PS1 double transgenic mice compared to C57BL/6J mice. In contrast, apoA1-ND treatment significantly reduced the impaired neuronal integrity and neuronal loss in APP/PS1 double transgenic mice.
Example 9: behavioral trace analysis of alzheimer model mice: morris Water maze experiment.
Purpose of the experiment: the spatial learning and memory ability of APP/PS1 double transgenic mice was evaluated.
The experimental method comprises the following steps: male APP/PS1 mice of 7 months of age were randomly divided into 2 groups, and injected with physiological saline or ApoA1-ND solution, respectively, in the tail vein for 4 weeks at 200 μ L per day. As a normal control group, 7-month-old male C57BL/6J mice were injected with physiological saline, and the mice were specifically grouped as follows.
| Group of | Test procedure |
| 1(Control) | C57BL/6J mice + intravenous saline for 4 |
| 2 | APP/PS1 mice + intravenous saline for 4 |
| 3 | APP/PS1 mice + intravenous ApoA1-ND (20. Mu.M) for 4 consecutive weeks |
The experiment is carried out for 5 days, and the first four days are positioning navigation experiments: the experiment begins by putting the mouse into a water pool (without a platform) to swim freely for 2min to enable the mouse to be familiar with the maze environment, putting the mouse on the platform for a plurality of seconds, and enabling the mouse to know that the escape platform exists in the water. And then, putting the mouse into a water pool from the surface wall of the quadrant (quadrant I) farthest from the platform, starting timing, stopping timing 5s after the mouse climbs the platform, allowing the swimming time to be 120s, recording the latency period as 120s if the mouse does not climb the platform 120s later, allowing the mouse to stand on the platform for a plurality of seconds, and finally wiping the mouse and putting the mouse into a cage. The mice were thus placed in the remaining three quadrants (i.e. the experiments were performed in sequence i, ii, iii, iv) in sequence, 4 quadrants per mouse per day for a total of 4 days. The training video analysis system automatically records the escape latency, the track (path), the total route, the total time, the average speed, the swimming distance of each quadrant, the time spent in each quadrant and the like when the underwater platform is reached. And (5) removing the original platform on the 5 th day, wherein the environment and the water temperature are the same as those of the positioning navigation test. The platform under the water surface is scattered, the mouse enters the water from the surface wall of the quadrant (quadrant I) farthest from the platform to carry out single test, and the rest quadrants do not need to be tested. The swimming trajectory of the mouse was recorded for 120s, after which the mouse was taken out and wiped dry water was put into the cage. The measuring indexes comprise the times of crossing the original platform (entering and leaving the platform), the percentage of the time of the quadrant on the original platform to the total time, the swimming distance of each quadrant and the like.
The experimental results are as follows: as can be seen in FIG. 9 (a), 7-month old male APP/PS1 mice received ApoA1-ND treatment. FIG. 9 (b) shows escape latency, which is significantly reduced in ApoA1-ND treated APP/PS1 double transgenic mice during the four-day water maze training. FIG. 9 (c) shows swimming speed, which is improved to some extent in the course of four-day water maze training for APP/PS1 double-transgenic mice treated by ApoA1-ND. FIG. 9 (d) shows the number of passes of each group of mice through the platform one day after platform removal, and it can be seen that APP/PS1 mice treated with ApoA1-ND pass the platform significantly more than APP/PS1 mice not treated with ApoA1-ND.
In summary, reference is made to fig. 9 (a), 9 (b), 9 (C) and 9 (d) in which they exhibit learning deficit compared to C57BL/6J mice. In contrast, apoA1-ND treated APP/PS1 dual transgenic mice showed significantly improved spatial learning and memory during the four-day water maze training, both in escape latency and swimming speed.
After the escape platform is dismantled, C57BL/6J mice intensively search the quadrant where the platform is located, and APP/PS1 double-transgenic mice treated by normal saline show an unfocused search strategy. After ApoA1-ND treatment, the search strategy of APP/PS1 double transgenic mice is significantly improved, has more positive exploratory performance, and occurs more frequently around the plateau position.
Example 10: evaluation experiment of biological safety of ApoA1-ND for treating AD.
The purpose of the experiment is as follows: evaluation of the biological safety of ApoA1-ND for the treatment of AD
The experimental method comprises the following steps:
male APP/PS1 mice of 7 months of age were randomly divided into 2 groups, and injected with physiological saline or ApoA1-ND solution, respectively, in the tail vein for 4 weeks at 200 μ L per day. As a normal control group, 7-month-old male C57BL/6J mice were injected with physiological saline, and the mice were specifically divided as follows.
| Group of | Test operation |
| 1(Control) | C57BL/6J mice + intravenous saline, continuous 4 |
| 2 | APP/PS1 mice + intravenous saline for 4 |
| 3 | APP/PS1 mice + intravenous ApoA1-ND (20. Mu.M) for 4 consecutive weeks |
Experimental mice were sacrificed and major organs were collected, fixed, dehydrated, embedded in paraffin, serially sectioned, hematoxylin and eosin stained, and finally evaluated by optical microscopy.
The experimental results are as follows:
there were no significant pathological changes in heart (A), liver (B), spleen (C) and kidney (E) in ApoA1-ND treated animals compared to saline treated animals. In the lung (D), however, inflammatory responses such as alveolar septal hemorrhage, perialveolar capillary congestion, and interstitial focal lymphocyte infiltration were reported in APP/PS1 mice treated with saline.
This pathological change was not seen in ApoA1-ND treated animals. Since HDL (high-density lipoprotein) has anti-inflammatory activity, it is speculated that ApoA1-ND might reduce the inflammatory response in the lung of APP/PS1 mice by the same mechanism.
As shown in FIG. 10, A, B, C, D and E correspond to heart, liver, spleen, lung and kidney, respectively. Treatment with ApoA1-ND reduced microglial proliferation and improved nervous system changes in APP/PS1 mice without additional significant brain tissue damage. Experimental data indicate that the in vivo use of ApoA1-ND is safe under current dosing regimens. Further evaluation of long-term in vivo safety has significant commercial value for anti-AD therapy.
The experimental result shows that ApoA1-ND has higher binding affinity to A beta monomers and oligomers, and the degradation of A beta in microglia and liver cells is accelerated. More importantly, the experimental result shows that ApoA1-ND can enter the brain through the blood brain barrier after the tail vein injection of the mouse.
After continuous tail vein injection treatment, the model mouse can reduce amyloid deposition, alleviate microglial cell proliferation, improve nervous system change and save memory defects.
Because the ApoA1-ND components are derived from protein and natural phospholipid, the protein system developed by the invention can efficiently pass through a blood brain barrier, has high brain targeting property, is safe and non-toxic, effectively eliminates Alzheimer's disease symptoms, and can be applied to preventing and treating early neurodegenerative diseases.
While the preferred embodiment and the corresponding examples of the present invention have been described, it should be understood that various changes and modifications, including but not limited to, adjustments of proportions, flows and amounts, which are within the scope of the invention, may be made by those skilled in the art without departing from the inventive concept thereof. While the preferred embodiment and the corresponding examples of the present invention have been described, it should be understood that various changes and modifications, including but not limited to, adjustments of proportions, flows and amounts, which are within the scope of the invention, may be made by those skilled in the art without departing from the inventive concept thereof.
SEQUENCE LISTING
<110> northwest university of industry
<120> protein phospholipid nano preparation, application and preparation method thereof
<130> 20220419
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Claims (10)
1. A nano preparation of protein phospholipid is characterized by being based on a protein phospholipid complex combined by ApoA1 protein and phospholipid;
wherein, the ApoA1 protein is ApoA1 protein without conserved sequence shown in SEQ ID No.1 or homologous sequence of the conserved sequence.
2. The nano-formulation of protein phospholipid of claim 1, wherein the amino acid sequence of the nano-formulation of protein phospholipid comprises:
one or two of 1 st amino acid sequence shown as SEQ ID No.2 and 2 nd amino acid sequence shown as SEQ ID No. 3.
3. The nano-formulation of protein phospholipid of claim 2, wherein the amino acid sequence of the nano-formulation of protein phospholipid further comprises:
the 3 rd amino acid sequence shown as SEQ ID No. 3.
4. The nano-preparation of protein phospholipid of claim 3 wherein the amino acid sequence of the nano-preparation of protein phospholipid is arranged sequentially from the 1 st amino acid sequence to the 2 nd amino acid sequence and from the 3 rd amino acid sequence.
5. The proteophospholipid nanoformulation of claim 1, wherein each nanoparticle of the proteopholipid nanoformulation has a diameter of 5-130nm.
6. The nano-formulation of protein phospholipid of claim 1, wherein the nano-formulation of protein phospholipid has an amount of embedded phospholipid of 100 to 1000 per nanoparticle.
7. Use of the nano preparation of protein phospholipid as defined in any one of claims 1-6 for the preparation of a product for the prevention and/or treatment of early neurodegenerative diseases.
8. Use of a nano-formulation of a proteolipid as defined in any of claims 1 to 6 for the preparation of a product for the treatment and/or prevention of CNS disorders.
9. A brain drug delivery system comprising the proteolipid nanoformulation according to any one of claims 1 to 6.
10. A method for preparing the nano preparation of the proteolipid as claimed in any of the claims 1 to 6, comprising:
taking ApoA1 protein, and obtaining a crude protein sample after induced expression;
purifying the crude protein sample to obtain purified protein;
and assembling the purified protein and dimyristoyl phosphatidylcholine to obtain the protein phospholipid nanometer preparation.
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